PROJECT SUMMARY Alcohol-associated liver disease (AALD) is a major and growing health concern with limited treatment options. The natural history of AALD (formerly known as alcoholic liver disease) includes fatty liver disease, alcoholic hepatitis and the development of fibrosis preceding end-stage cirrhosis. MicroRNAs (miRNAs) represent a new class of therapeutics due to their ability to simultaneously affect multiple fibrosis-associated pathways. Among possible targets, miR-155 is involved in inflammatory responses mediated by Kupffer cells (KCs) that affect fibrogenic events in multiple other hepatic cells. Chemokine receptor CXCR4 and its cognate ligand stromal cell- derived factor-1 play important and complex roles in the pathogenesis of AALD, including the coordination of the initial immune reaction upon liver injury and later in controlling the progression of liver fibrosis through its activating effect on hepatic stellate cells (HSCs) and collagen production. The goal of this project is to develop integrated miRNA delivery platform based on self-assembled nanoparticles (polyplexes) that deliver anti-miR- 155 to activated KCs and in parallel inhibit CXCR4 signaling in activated HSCs in the liver. The delivery platform is based on innovative CXCR4 inhibitors based on cyclam-modified low molecular weight poly(ethylenimine)s (C-PEI) that efficiently encapsulate and systemically deliver miRNA. The objective is to test the hypothesis that C-PEI/miRNA will lead to enhanced combination effect due to attenuation of profibrogenic signaling of both hepatic macrophages and the matrix-producing HSCs. We will accomplish the overall objectives in three specific aims. In Aim 1, we will optimize formulation of the C-PEI-miRNA nanoparticles that deliver anti-miR-155 in liver fibrosis. Based on encouraging antifibrotic activity in our preliminary studies, we hypothesize that polyplex modification with mannose and with stabilizing cholesterol moieties will result in efficient delivery of the miRNA to activated KCs, while excess free C-PEI will target activated HSCs. In Aim 2, we will test the in vivo therapeutic efficacy of the polyplexes in the bile-duct ligation (BDL) model and mouse models of chronic alcohol administration with exposure to CCl4. The goal is to conduct comprehensive evaluation of therapeutic efficacy of the optimized C-PEI-Chol/anti-miR-155 polyplexes in different models and multiple stages of fibrosis. The findings from the mouse models will be validated in cultured human precision-cut liver slices. In Aim 3, we will determine the mechanism of antifibrotic activity of the combined miR-155 and CXCR4 inhibition. Although our preliminary data are consistent with miR-155 downregulation in KCs and inhibition of CXCR4 in activated HSCs, the precise mechanism of action of the C-PEI/anti-miR-155 polyplexes is not known. Therefore, the studies in this aim will be designed to ascertain the underlying mechanisms of action of the polyplexes. Overall, this project will contribute to the fundamental understanding of the role and therapeutic potential of CXCR4 and miR-155 inhibition in AALD and will innovatively address targeted delivery of drug/miRNA combinations as an anti-fibrotic treatment.